Studies on the enzyme adenylate cyclase have in general emphasized hormonal regulation of the enzyme. Such studies have indicated that adenylate cyclase consists of at least 3 components: receptors for stimulatory and inhibitory ligands, guanyl nucleotide-binding proteins (Gs and Gi), and a catalytic unit (C). Activators of adenylate cyclase which do not act through specific receptors (Gpp(NH)p, GTP, Fl-, calmodulin) have been useful in clarifying how each of these components function to stimulate or attenuate cellular cAMP synthesis. The diterpene forskolin is a potent activator of adenylate cyclase, and it seemed appropriate to clarify its mode of activation. Our approach to studying forskolin activation was to solubilize bovine brain membranes with CHAPS, separate the C and Gs components of adenylate cyclase, and study the effect of forskolin on the separate components. We found that forskolin activates C about 300%; half-maximal activation occurs at 5 MuM forskolin. C was also activated by calmodulin and by phospholipids; the effects of maximally activating concentrations of these activators were additive to those of forskolin at all concentrations of the latter. When samples of C were mixed with column fractions that contained Gs, activation by Gpp(NH)p was demonstrable. In this system, Gpp(NH)p activation was additive to forskolin-stimulated activity. Prior incubation of C with either forskolin or Gs did not potentiate the ability of C to respond to the other agent. It is concluded that the locus of action of forskolin on brain adenylate cyclase is at the catalytic unit; forskolin does not appear to alter the interaction of Gs with C.